The proposed research is designed to study the regulation of acetate metabolism and unsaturated fatty acid biosynthesis using E. coli as a model system. Growth of this organism on substrates such as acetate or fatty acids requires the operation of the glyoxylate shunt. The two unique enzymes of the glyoxylate shunt, isocitrate lyase and malate synthase A, are induced when acetate or fatty acids are present in the growth media. The structural genes for isocitrate lyase, aceA, and malate synthase A, aceB, map at 90 min on the E. coli linkage map and appear to comprise an operon. Expression of this ace operon is under the transcriptional control of two regulatory genes, icLR and fadR. At present, our studies suggest that the iclR and fadR gene products act independently to repress the ace operon. In order to understand the molecular details of the mechanism(s) by which the icLR and fadR gene products regulate the ace operon, we plan to (1) clone the iclR, fadR, and ace operon genes, (2) purify and characterize the icLR and fadR gene products, and (3) determine in vitro if the fadR and iclR gene products bind plasmids carrying the cloned ace operon genes. We have recently found that a functional fadR gene is required in order for E. coli to optimally synthesize unsaturated fatty acids (UFA). The two enzymes known to be involved specifically with the biosynthesis of UFA, are Beta-hydroxydecanoyl thioester dehydrase and BETA -ketoacyl-acyl carrier protein synthase I. In order to understand the role the fadR gene plays in UFA biosynthesis, we plan to (1) compare the level of the two UFA biosynthetic enzymes in fadR and fadR+ strains, (2) determine if the fadR gene regulated the transcription of the genes that code for the two UFA biosynthetic enzymes, (3) clone the structural genes coding for the UFA biosynthetic enzymes, and (4) determine if the fadR gene binds to plasmid clones of the two UFA biosynthetic enzymes.